The mitochondrial genome of Tipula (Vestiplex) aestiva Savchenko, 1960 (Diptera: Tipulidae)

Abstract The subgenus Vestiplex of the genus Tipula is one of the most species-rich groups in the family Tipulidae. Here, we present the first mitochondrial genome of the subgenus Vestiplex. The nearly complete mitochondrial genome of Tipula aestiva Savchenko, 1960 (Genbank accession number: OM287601) was 16083 bp in length and consisted of 13 PCGs, 22 tRNA and two rRNA genes. The phylogenetic the tree of family Tipulidae was reconstructed based on 13 PCGs sequences using the maximum likelihood method. The result strongly supported the monophyly of the family Tipulidae. The subgenus Vestiplex is indicated as the sister group of subgenera Pterelachisus and Formotipula.


Introduction
The genus Tipula is one of the most diverse groups in the family Tipulidae with over 40 subgenera and 2400 species, which are distributed worldwide (Oosterbroek 2022). However, the monophyly of the genus has long been a controversial issue. Since the mitochondrial (mt) genomes were introduced into phylogenetic studies, several Tipula species of different subgenera had their mitogenomes sequenced and published, including those of subgenera Acutipula, Dendrotipula, Formotipula , Lunatipula, Nippotipula (Beckenbach 2011), Pterelachisus, and Yamatotipula (Zhao et al. 2019). As one of the largest subgenera of the genus Tipula, Vestiplex contains over 180 described species worldwide, but none of which had its mitogenome available. Adults of Vestiplex usually occur in forests or grasslands, while their larvae were considered to be saprophagous (Gelhaus 1986). Adults can generally be characterized by the medium-sized body with the patterned wings and the ninth tergite with a shallow concavity and sclerotized saucer or completely divided longitudinally by the pale membrane (Alexander 1935;Alexander and Byers 1981) and larvae can generally be characterized by the dark band separating anus from anal papillae, in addition to marginal band encircling the anal area (Gelhaus 1986). Here, we presented the nearly complete mitochondrial genome of Tipula aestiva Savchenko, 1960, which is the first mitochondrial genome of the subgenus Vestiplex. The results provide valuable information for future phylogenetic and evolutionary studies.

Materials and methods
The specimen of Tipula (Vestiplex) aestiva Savchenko, 1960 (Voucher number: CAUGS20200802) were collected by Shang Gao on 2 August 2020 from Zhamashixigou (38.2139 N, 100.0368E, 3048 m), Qilian, Qinghai Province, China. It was preserved in 95% ethanol and deposited in the Entomological Museum of China Agricultural University, Beijing (Liang Wang, 1352659341@qq.com). The specimen were identified by Yuetian Gao based on the following morphological characteristics: flagellomeres with verticils shorter than half of corresponding segments; tergite 9 in the shape of the slightly concaved saucer, with the elevated anterior border (Starkevich et al. 2019). Whole genomic DNA was extracted from the thorax of a single specimen using QIAamp DNA Blood Mini Kit (Qiagen, Germany). Then, the DNA concentration was measured by Agilent 5400. The sequencing library was generated using NEB NextV R Ultra TM DNA Library Prep Kit. The sample was subsequently sequenced paired-end on one lane with Illumina NovaSeq 6000, with the desired insert size of 350 bp and read length of 150 bp. Approximately 6 G of raw data was produced by sequencing. The processes above were accomplished by the Novogene biotechnology company (Beijing). The mitochondrial genome was assembled and annotated by MitoZ (v.2.3).
And the start and stop codons of each protein-coding gene (PCG) were checked manually in Geneious (v. 9.0.2).

Results
The nearly complete mitogenome of Tipula (Vestiplex) aestiva was 16,083 bp in length (Genbank accession number: OM287601), including 13 protein-coding genes, 22 tRNA genes and 2 rRNA genes (12S rRNA and 16S rRNA). The arrangement of all mitochondrial genes matched other published mitogenomes of Tipulidae species Beckenbach 2011;Zhao et al. 2019). The control region was incompletely obtained in this research. The nucleotide composition of the mitogenome was biased toward A and T, with 77.4% of A þ T content (A ¼ 38.9%, T ¼ 38.5%, G ¼ 8.9%, C ¼ 13.7%). Most PCG genes start with ATT, ATG or ATA codons, except that three genes (COI, NAD5 and NAD1) start with TCG, CAC, CAA codons, respectively. Most PCG genes stop with TAA, TAG or TAT codons, but NAD4L stops with ATT codons. Two PCG genes stopping with A þ tRNA are NAD5 and NAD4. COX2 stops with T þ tRNA.
In our research, the maximum likelihood (ML) analysis was conducted based on 13 PCGs from 9 species of the family Tipulidae (Ren et al. 2019b), and Limonia phragmitidis (Ren et al. 2019a) was chosen as outgroup using RAxML 8.2.12 (Stamatakis 2006). Our results support that the family Tipulidae is monophyletic (Figure 1). The monophyly of the genus Tipula was not supported, which is consistent with the phylogenetic result of the previous research (Petersen et al. 2010). The genus Nephrotoma is the sister group of subgenus Acutipula. The subgenus Vestiplex is the sister group of subgenus Pterelachisus and subgenus Formotipula, which is similar to the proposal of Savchenko (1979